Cysteine Residues in the Major Capsid Protein,Vp1, of the JC Virus Are Important for Protein Stability and Oligomer Formation

The capsid of the human polyomavirus JC virus (JCV) consists of 72 pentameric capsomeres of a major structural protein, Vp1. The cysteine residues of the related Vp1 of SV40 are known to contribute to Vp1 folding, pentamer formation, pentamer-pentamer contacts, and capsid stabilization. In light of the presence of a slight structural difference between JCV Vp1 and SV40 counterpart, the way the former folds could be either different from or similar to the latter. We found a difference: an important contribution of Vp1 cysteines to the formation of infectious virions, unique in JCV and absent in SV40. Having introduced amino acid substitution at each of six cysteines (C42, C80, C97, C200, C247, and C260) in JCV Vp1, we found that, when expressed in HeLa cells, the Vp1 level was decreased in C80A and C247A mutants, and remained normal in the other mutants. Additionally, the C80A and C247A Vp1-expressing cell extracts did not show the hemagglutination activity characteristic of JCV particles. The C80A and C247A mutant Vp1s were found to be less stable than the wild-type Vp1 in HeLa cells. When produced in a reconstituted in vitro protein translation system, these two mutant proteins were stable, suggesting that some cellular factors were responsible for their degradation. As determined by their sucrose gradient sedimentation profiles, in vitro translated C247A Vp1 formed pentamers, but in vitro translated C80A Vp1 was entirely monomeric. When individually incorporated into the JCV genome, the C80A and C247A mutants, but not the other Vp1 cysteine residues mutants, interfered with JCV infectivity. Furthermore, the C80A, but not the C247A, mutation prevented the nuclear localization of Vp1 in JCV genome transfected cells. These findings suggest that C80 of JCV Vp1 is required for Vp1 stability and pentamer formation, and C247 is involved in capsid assembly in the nucleus.     

Molecular Markers for Granulovacuolar Degeneration Are Present in Rimmed Vacuoles

Background

Rimmed vacuoles (RVs) are round-oval cytoplasmic inclusions, detected in muscle cells of patients with myopathies, such as inclusion body myositis (IBM) and distal myopathy with RVs (DMRV). Granulovacuolar degeneration (GVD) bodies are spherical vacuoles containing argentophilic and hematoxyphilic granules, and are one of the pathological hallmarks commonly found in hippocampal pyramidal neurons of patients with aging-related neurodegenerative diseases, such as Alzheimer''s disease and Parkinson''s disease. These diseases are common in the elderly and share some pathological features. Therefore, we hypothesized that mechanisms of vacuolar formation in RVs and GVD bodies are common despite their role in two differing pathologies. We explored the components of RVs by immunohistochemistry, using antibodies for GVD markers.

Methods

Subjects included one AD case, eight cases of sporadic IBM, and three cases of DMRV. We compared immunoreactivity and staining patterns for GVD markers. These markers included: (1) tau-modifying proteins (caspase 3, cyclin-dependent kinase 5 [CDK5], casein kinase 1δ [CK1δ], and c-jun N-terminal kinase [JNK]), (2) lipid raft-associated materials (annexin 2, leucine-rich repeat kinase 2 [LRRK2], and flotillin-1), and (3) other markers (charged multi-vesicular body protein 2B [CHMP2B] and phosphorylated transactive response DNA binding protein-43 [pTDP43]) in both GVD bodies and RVs. Furthermore, we performed double staining of each GVD marker with pTDP43 to verify the co-localization.

Results

GVD markers, including lipid raft-associated proteins and tau kinases, were detected in RVs. CHMP2B, pTDP43, caspase 3, LRRK2, annexin 2 and flotillin-1 were detected on the rim and were diffusely distributed in the cytoplasm of RV-positive fibers. CDK5, CK1δ and JNK were detected only on the rim. In double staining experiments, all GVD markers colocalized with pTDP43 in RVs.

Conclusions

These results suggest that RVs of muscle cells and GVD bodies of neurons share a number of molecules, such as raft-related proteins and tau-modifying proteins.     

Glucagon-Like Peptide-1 Secretory Function as an Independent Determinant of Blood Pressure: Analysis in the Tanno-Sobetsu Study

Aims

Roles of glucagon-like peptide-1 (GLP-1) in extra-pancreatic tissues remain unclear. The aim of this study was to examine determinants of GLP-1 secretory function and possible contribution of GLP-1 to blood pressure (BP) regulation.

Methods and Results

We recruited 128 subjects who received annual examinations and 75g-oral glucose tolerance tests (OGTT) in the Tanno-Sobetsu cohort. Subjects on regular medications for cardiovascular and/or metabolic diseases were excluded, and data for the remaining 103 subjects were used for the univariate and multivariate analyses. Age, plasma glucose (PG), hemoglobin A1c (HbA1c), plasma insulin, and serum lipids were not selected as independent determinants of fasting GLP-1 level by multiple linear regression analysis. However, age and female sex were selected as independent positive determinants of the area under the curve of GLP-1 level during OGTT (AUC_GLP-1), an index of GLP-1 secretory function. Multiple linear regression analysis indicated that AUC_GLP-1 was an independent negative predictor of systolic BP (SBP), while AUC_GLP-1 was not correlated with fasting PG or HbA1c level. In subgroup analyses using the median of AUC_GLP-1 to divide the study subjects into high and low GLP-1 response groups, AUC_GLP-1 was significantly correlated with both SBP and diastolic BP (r = 0.40 and 0.28, respectively) in the low GLP-1 response group but not in the high GLP-1 response group.

Conclusions

The results of the present study suggest that GLP-1 secretory function is involved in prevention of BP elevation and that the GLP-1 response to oral glucose rather increases with aging perhaps as an adaptive phenomenon.     

Circulating Levels of Fatty Acid-Binding Protein Family and Metabolic Phenotype in the General Population

Objective

Fatty acid-binding proteins (FABPs) are a family of 14-15-kDa proteins, and some FABPs have been to be used as biomarkers of tissue injury by leak from cells. However, recent studies have shown that FABPs can be secreted from cells into circulation. Here we examined determinants and roles of circulating FABPs in a general population.

Methods

From the database of the Tanno-Sobetsu Study, a study with a population-based cohort design, data in 2011 for 296 subjects on no medication were retrieved, and FABP1∼5 in their serum samples were assayed.

Results

Level of FABP4, but not the other isoforms, showed a gender difference, being higher in females than in males. Levels of all FABPs were negatively correlated with estimated glomerular filtration rate (eGFR), but a distinct pattern of correlation with other clinical parameters was observed for each FABP isoform; significant correlates were alanine aminotransferase (ALT), blood pressure (BP), and brain natriuretic peptide (BNP) for FABP1, none besides eGFR for FABP2, age, BP, and BNP for FABP3, age, waist circumference (WC), BP, BNP, lipid variables, high-sensitivity C-reactive protein (hsCRP), and HOMA-R for FABP4, and age, WC, BP, ALT, BNP, and HOMA-R for FABP5. FABP4 is the most strongly related to metabolic markers among FABPs. In a multivariate regression analysis, FABP4 level was an independent predictor of HOMA-R after adjustment of age, gender, WC, BP, HDL cholesterol, and hsCRP.

Conclusions

Each FABP isoform level showed a distinct pattern of correlation with clinical parameters, although levels of all FABPs were negatively determined by renal function. Circulating FABP4 appears to be a useful biomarker for detecting pre-clinical stage of metabolic syndrome, especially insulin resistance, in the general population.     

Urinary Fetuin-A Is a Novel Marker for Diabetic Nephropathy in Type 2 Diabetes Identified by Lectin Microarray

We analyzed the urine samples of patients with type 2 diabetes at various stages of diabetic nephropathy by lectin microarray to identify a biomarker to predict the progression of diabetic nephropathy. Japanese patients with type 2 diabetes at various stages of nephropathy were enrolled and we performed lectin microarray analyses (n = 17) and measured urinary excretion of fetuin-A (n = 85). The increased signals of urine samples were observed in Siaα2-6Gal/GalNAc-binding lectins (SNA, SSA, TJA-I) during the progression of diabetic nephropathy. We next isolated sialylated glycoproteins by using SSA-lectin affinity chromatography and identified fetuin-A by liquid chromatography–tandem mass spectrometer. Urinary excretion of fetuin-A significantly increased during the progression of albuminuria (A1, 0.40±0.43; A2, 0.60±0.53; A3 1.57±1.13 ng/gCr; p = 7.29×10⁻⁸) and of GFR stages (G1, 0.39±0.39; G2, 0.49±0.45; G3, 1.25±1.18; G4, 1.34±0.80 ng/gCr; p = 3.89×10⁻⁴). Multivariate logistic regression analysis was employed to assess fetuin-A as a risk for diabetic nephropathy with microalbuminuria or GFR<60 mL/min. Fetuin-A is demonstrated as a risk factor for both microalbuminuria and reduction of GFR in diabetic nephropathy with the odds ratio of 4.721 (1.881–11.844) and 3.739 (1.785–7.841), respectively. Collectively, the glycan profiling analysis is useful method to identify the urine biomarkers and fetuin-A is a candidate to predict the progression of diabetic nephropathy.     

Metabolome analysis of food-chain between plants and insects

Evolution has shown the co-dependency between host plants and predators (insects), especially inevitable dependency of predators on plant biomass for securing their energy sources. It was postulated that NAD⁺ source used for major energy producing pathway is the glycerol-3-phosphate shuttle in insects. Using high throughput metabolomics approach, we found that larva of leaf beetle (Gastrophysa atrocyanea), which feed oxalate-rich plants (Rumex obtusifolius), possessed a unique mechanism for accumulating unusually high amounts of lactate. Similarly, larva of butterfly (Papilio machaon) fed with fennel (Foeniculum vulgare) accumulated lactate. Same butterfly also showed the elevated level of glycerol-3-phosphate equivalent to lactate. These evidences provide new insights into the mechanism underlying metabolite alteration between host plants and insect herbivores.     

The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.     

Detection of Ancestry Informative HLA Alleles Confirms the Admixed Origins of Japanese Population

The polymorphisms in the human leukocyte antigen (HLA) region are powerful tool for studying human evolutionary processes. We investigated genetic structure of Japanese by using five-locus HLA genotypes (HLA-A, -B, -C, -DRB1, and -DPB1) of 2,005 individuals from 10 regions of Japan. We found a significant level of population substructure in Japanese; particularly the differentiation between Okinawa Island and mainland Japanese. By using a plot of the principal component scores, we identified ancestry informative alleles associated with the underlying population substructure. We examined extent of linkage disequilibrium (LD) between pairs of HLA alleles on the haplotypes that were differentiated among regions. The LDs were strong and weak for pairs of HLA alleles characterized by low and high frequencies in Okinawa Island, respectively. The five-locus haplotypes whose alleles exhibit strong LD were unique to Japanese and South Korean, suggesting that these haplotypes had been recently derived from the Korean Peninsula. The alleles characterized by high frequency in Japanese compared to South Korean formed segmented three-locus haplotype that was commonly found in Aleuts, Eskimos, and North- and Meso-Americans but not observed in Korean and Chinese. The serologically equivalent haplotype was found in Orchid Island in Taiwan, Mongol, Siberia, and Arctic regions. It suggests that early Japanese who existed prior to the migration wave from the Korean Peninsula shared ancestry with northern Asian who moved to the New World via the Bering Strait land bridge. These results may support the admixture model for peopling of Japanese Archipelago.